It is estimated that exposure to tobacco smoke, either directly, or through second hand smoke affects more than one third of the world's population, and according to the World Health Organization, tobacco kills almost 6 million people annually. In addition to its enormous health consequences, recent and emerging data indicate that tobacco smoke exposure may have transgenerational consequences. For example, children of men who smoked prior to conception have increased risk of developing childhood leukemia. This indicates that smoking can cause heritable genetic and/or epigenetic changes in the gametes that can negatively impact offspring health. From pilot studies we know that global sperm DNA methylation levels are significantly reduced compared with non-smokers, and multiple studies have demonstrated that tobaccos smoke exposure changes methylation patterns in somatic tissues. For this study we propose to fully characterize the effect of tobacco smoke exposure on sperm DNA methylation patterns in the mouse. We will perform breeding experiments to follow the observed changes in offspring across two generations. In addition, as tobacco smoke induced methylation changes are postulated to occur in through oxidative stress pathways, we will use a mouse model in which a major antioxidant response pathway (Nrf2) has been disrupted to determine whether this gene disruption amplifies the epigenetic changes that occur due to tobacco smoke exposure as well as the transgenerational consequences of that effect. Lastly, we will use a methylation microarray to assess the effects of tobacco smoke exposure on the sperm methylome in 150 men who smoke compared with 150 non- smokers. These experiments are a critical step in understanding the potential long term, transgenerational effects of tobacco smoke exposure and will lend insight into the mechanisms for exposure-induced methylation perturbations. The human studies proposed in Aim 3 will lend insight into the relevance of animal studies for paternally mediated, transgenerational inheritance in humans.